Faculty Bibliography

Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed. J. Cell. Biochem. 112: 607-613, 2011. (c) 2010 Wiley-Liss, Inc

Skeletogenesis depends on the activity of bone-forming cells derived from mesenchymal cells. The pathways that control mesenchymal cell differentiation are not well understood. We propose that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblasts. In mouse embryos, Foxo1 expression is higher in skeletal tissues, while Foxo1 silencing has a drastic impact on skeletogenesis and craniofacial development, specially affecting pre-maxilla, nasal bone, mandible, tibia, and clavicle. Similarly, Foxo1 activity and expression increase in mouse mesenchymal cells under the influence of osteogenic stimulants. In addition, silencing Foxo1 blocks the expression of osteogenic markers such as Runx2, alkaline phosphatase, and osteocalcin and results in decreased culture calcification even in the presence of strong osteogenic stimulants. Conversely, the expression of these markers increases significantly in response to Foxo1 overexpression. One mechanism through which Foxo1 affects mesenchymal cell differentiation into osteoblasts is through regulation of a key osteogenic transcription factor, Runx2. Indeed, our results show that Foxo1 directly interacts with the promoter of Runx2 and regulates its expression. Using a tibia organ culture model, we confirmed that silencing Foxo1 decreases the expression of Runx2 and impairs bone formation. Furthermore, our data reveals that Runx2 and Foxo1 interact with each other and cooperate in the transcriptional regulation of osteoblast markers. In conclusion, our in vitro, ex vivo, and in vivo results strongly support the notion that Foxo1 is an early molecular regulator in the differentiation of mesenchymal cells into osteoblast.

This study examines the role of F-spondin, an extracellular matrix protein of osteoarthritic cartilage, during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. Functional studies using tibial organ cultures indicated that F-spondin inhibited ( approximately 35%, p = 0.02), and antibodies to F-spondin increased ( approximately 30%, p < 0.1) longitudinal limb growth relative to untreated controls. In cell cultures, induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-beta treatment led to a significant upregulation of F-spondin (p < 0.05). F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels (p < 0.05), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin (TSR) domain inhibited RA induced AP activity 40% compared to controls (p < 0.05). The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-beta from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-beta dependent pathways.

It has been shown that inhibiting the expression of certain cytokines decreases the rate of tooth movement. Here, we hypothesized that stimulating the expression of inflammatory cytokines, through small perforations of cortical bone, increases the rate of bone remodeling and tooth movement. Forty-eight rats were divided into 4 groups: 50-cN force applied to the maxillary first molar (O), force application plus soft tissue flap (OF), force application plus flap plus 3 small perforations of the cortical plate (OFP), and a control group (C). From the 92 cytokines studied, the expression of 37 cytokines increased significantly in all experimental groups, with 21 cytokines showing the highest levels in the OFP group. After 28 days, micro-computed tomography, light and fluorescent microscopy, and immunohistochemistry demonstrated higher numbers of osteoclasts and bone remodeling activity in the OFP group, accompanied by generalized osteoporosity and increased rate of tooth movement

Bone repair and regeneration is one of the most extensively studied areas in the field of tissue engineering. All of the current tissue engineering approaches to create bone focus on intramembranous ossification, ignoring the other mechanism of bone formation, endochondral ossification. We propose to create a transient cartilage template in vitro, which could serve as an intermediate for bone formation by the endochondral mechanism once implanted in vivo. The goals of the study are (1) to prepare and characterize type I collagen sponges as a scaffold for the cartilage template, and (2) to establish a method of culturing chondrocytes in type I collagen sponges and induce cell maturation. Collagen sponges were generated from a 1% solution of type I collagen using a freeze/dry technique followed by UV light crosslinking. Chondrocytes isolated from two locations in chick embryo sterna were cultured in these sponges and treated with retinoic acid to induce chondrocyte maturation and extracellular matrix deposition. Material strength testing as well as microscopic and biochemical analyzes were conducted to evaluate the properties of sponges and cell behavior during the culture period. We found that our collagen sponges presented improved stiffness and supported chondrocyte attachment and proliferation. Cells underwent maturation, depositing an abundant extracellular matrix throughout the scaffold, expressing high levels of type X collagen, type I collagen and alkaline phosphatase. These results demonstrate that we have created a transient cartilage template with potential to direct endochondral bone formation after implantation.

This study evaluated the effect of a bioactive ceramic coating, in the nanothickness range, onto a moderately rough surface on the osteogenic behavior of human bone cells. The cells were harvested from the mandibular mental region and were cultured over Ti-6Al-4V disks of different surfaces: as-machined (M), alumina-blasted/acid etched (AB/AE), and alumina-blasted/acid-etched + 300-500 nm thickness amorphous Ca- and P-based coating obtained by ion beam-assisted deposition (Nano). The culture was then evaluated regarding cell viability, adhesion, morphology, immunolocalization of osteopontin (OPN) and alkaline phosphatase (ALP). The results showed that the surface treatment did not interfere with cell viability. At 1 day, AB/AE and Nano showed higher adhesion than the M surface (p < 0.001). Higher adhesion was observed for the M than the Nano surface at 7 days (p < 0.005). The percentage of cells showing intracellular labeling for OPN at day 1 was significantly higher for the Nano compared to M surface (p < 0.03). The percentage of ALP intracellular labeling at 7 days was significantly higher for the AB/AE compared to the M surface (p < 0.0065); no differences were detected at 14 days. Our results suggest that the presence of a thin bioactive ceramic coating on a rough substrate did not favor the events related to in vitro osteogenesis. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Current approaches for immediately loading dental implants rely upon clinical trials and evidence-based practice. Despite their wide utility, these studies have limited predictability because they ignore a whole set of parameters, mainly the environmental parameters that have increasingly influenced the bone biological response. Bone is a very dynamic tissue and its reaction to surgical and functional implant procedures is variable; therefore, bone remodeling must be a key factor in any clinical decision, and a need for reliable tests to evaluate this process is an eventual challenge to implant success. New advances in medicine based upon individual genomic, proteomic and metabolomic studies incorporate the impact of environmental elements, permitting a better targeting of implant therapy. Previously, we proposed a new clinical concept of implant therapy based on personalized bone turnover. Here, we elaborate on current tests and future 'omics' biotechnologies to assess the turnover process, hence, providing a realistic approach to individual evaluations of bone remodeling

Chitosan scaffolds have been shown to possess biological and mechanical properties suitable for tissue engineering and clinical applications. In the present work, chitosan sponges were evaluated regarding their ability to support cartilage cell proliferation and maturation, which are the first steps in endochondral bone formation. Chitosan sponges were seeded with chondrocytes isolated from chicken embryo sterna. Chondrocyte/chitosan constructs were cultured for 20 days, and treated with retinoic acid (RA) to induce chondrocyte maturation and matrix synthesis. At different time points, samples were collected for microscopic, histological, biochemical, and mechanical analyses. Results show chondrocyte attachment, proliferation, and abundant matrix synthesis, completely obliterating the pores of the sponges. RA treatment caused chondrocyte hypertrophy, characterized by the presence of type X collagen in the extracellular matrix and increased alkaline phosphatase activity. In addition, hypertrophy markedly changed the mechanical properties of the chondrocyte/chitosan constructs. In conclusion, we have developed chitosan sponges with adequate pore structure and mechanical properties to serve as a support for hypertrophic chondrocytes. In parallel studies, we have evaluated the ability of this mature cartilage scaffold to induce endochondral ossification.